247
Brain Research, 549 (1991) 247-252 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116613F BRES 16613
Dorsal medullary injection of atrial natriuretic factor (ANF) excites vagal efferents and inhibits gastric motility Monica J. McCann, Kathy Nice-Lepard and Richard C. Rogers Department of Physiology and Neuroscience Program, Ohio State University College of Medicine, Columbus, OH 43210 (U.S.A.) (Accepted 11 December 1990)
Key words: Atrial natriuretic factor; Medulla oblongata; Gastric motility; Efferent vagus; Extracellular recording
Atrial natriuretic factor (ANF)-immunoreactive fibers are found in the dorsal motor nucleus of the vagus (DMN) along with receptors for that peptide. Previous investigations showed that ANF injections into the DMN did not influence cardiovascular functions. Since the DMN is largely (but not exclusively) involved with the control of gastrointestinal functions, we hypothesized that ANF may act on gastric, rather than cardiovascular vagal efferents. Injections of ANF (20 pmol rat atriopeptin III in 20 nl) into the DMN evoked a vagally dependent reduction in gastric motility. In a separate electrophysiologicai study, 10 of 15 (66%) antidromically identified DMN neurons were excited by micropressure-applied ANF (25-500 fmol in 50-1000 pl). We propose that ANF-containing neurons in the DMN reduce gastric motility by activating vagal efferents that synapse with inhibitory neurons in the gastric enteric nervous system. INTRODUCTION
using a combination of micropressure injection and extracellular recording techniques.
Atrial natriuretic factor ( A N F ) , which was first isolated from extracts of rat atria, is known to have n u m e r o u s effects on cardiovascular function and fluid homeostasis when administered into the peripheral circulation2"5-7. Immunocytochemical studies report that A N F is also present within n e u r o n s in the central nervous system ~6'3°'32'33. The role(s) of A N F in the CNS remains to be elucidated, however it is interesting to note that ANF-like immunoreactive fibers and high affinity A N F binding sites are found in the nucleus of the solitary tract (NTS) and the dorsal motor nucleus (DMN) 16"27'32,33. This suggests that A N F may influence the vagal control of the heart and/or other vagally innervated organs. Indeed, several investigators have reported that A N F injections into the NTS provoke a modest (5%) cardioacceleration while similar injections into the D M N have no effect on cardiovascular function8,22. Because the D M N also is involved in the parasympathetic control of digestive functions H.18,26, we hypothesized that A N F may influence vagal efferent outflow to the stomach. We investigated this hypothesis by measuring the effect of A N F injections into the D M N on gastric corpus/antrum motility as well as heart rate in the rat. Furthermore, in a second set of experiments, we determined whether A N F influenced the firing rate of single D M N neurons
MATERIALS AND METHODS
ANF/gastric motility studies Male Long-Evans rats (250-450 g) were anesthetized with urethane (ethylcarbamate, 1.2 g/kg). The trachea was catheterized to maintain an open airway. Then, a laparotomy was performed and a miniature strain gauge (RB Products, 153 Nautilus Dr. Madison, WI) was sewn in parallel with corpus/antral circular smooth muscle to measure gastric motility and tone. After closing the incision, the animal was mounted in a stereotaxic frame, and the dorsal medulla was exposed. The tips of combination metal microstimulation/ double barrelled micropipette arrays2s were placed in the medial portion of the DMN. One pipette barrel was filled with ANF (rat atriopeptin III; ANF 5-28, Sigma, St. Louis, MO) dissolved in isotonic saline while the other barrel contained only the saline solution. Both pipette barrels were connected to a micropressure injection system (Medical Systems BH2, Great Neck, NY). The metal electrode was used to determine when the electrode array was positioned in the DMN. Briefly, electrical pulses (25/tA, 0.5 ms duration, 10 Hz, 2-5 s) were generated by a conventional stimulator and delivered to the electrode through a constant-current stimulus isolation unit; this stimulus results in a reduction in heart rate and gastric motility when the tip is positioned in the DMN29. The location of the pipette tips were confirmed by passing 50 pA cathodal d.c. through the stimulating electrode. Lesions were subsequently located by examining the brainstem histologically. To measure gastric motility the leads from the strain gauge were connected to a Wheatstone bridge-based strain gauge amplifier of conventional design. The output of this amplifier was directed simultaneously to a Grass polygraph and to the A-to-D convertor inputs of an RC Electronics waveform storage/analysis system. In
Correspondence: M.J. McCann, Department of Physiology, Ohio State University College of Medicine, 333 W. 10th Ave., Columbus, OH 43210, U.S.A.
248 this way, gastric motility data could be displayed in real time while being digitized for subsequent analysis or graphic display. To measure the heart rate, needle electrodes were placed over the thorax and hindlimb to record the ECG. ECG signals were used to produce a continuous plot of heart rate vs time using a ratemeter circuit connected to another channel of the polygraph. While monitoring motility and heart rate, either saline (20 nl) or ANF (20 pmol in 20 nl) was injected into the DMN (n = 7 rats). The amount of solution ejected from the pipettes was measured directly by observing the meniscus of the ejectate in the pipette with a 175 × long-working distance microscope (Melles Griot, Irvine, CA). Each injection was separated in time by at least 20 min. In an additional group of 4 rats, suture loops were placed around the vagal trunks after catheterizing the trachea. Thirty min prior to the administration of ANF into the DMN, the vagus nerves were severed by pulling the suture loops. All motility data were scored by applying the minute motility index 25 for a period of 5 min before and 5 min after DMN injections of saline or ANE Non-parametric statistics (Wilcoxon matched pairs test) were applied to the differences in motility indices between saline and ANF treatments.
A N F = 23.3 + 2.0 ( S . E . M . ) ; i n d e x after A N F = 22.0 _+ 2.5, P > 0.05; see Fig. l c and d). In c o n t r a s t to gastric motility, h e a r t rate was not a l t e r e d by A N F injections c e n t e r e d on the D M N
(data not shown).
Histological
analysis c o n f i r m e d the p o s i t i o n of the p i p e t t e tips within the D M N . In the s e c o n d series of e x p e r i m e n t s , A N F was microp r e s s u r e - i n j e c t e d on D M N n e u r o n s that w e r e identified using e l e c t r o p h y s i o l o g i c a l criteria. O f 28 a n t i d r o m i c a i l y a c t i v a t e d cells e n c o u n t e r e d , 15 cells w e r e r e c o r d e d for a sufficient length o f t i m e to d e t e r m i n e its r e s p o n s e to i n j e c t i o n of saline v e h i c l e and A N E O f t h e s e 15 cells, 10
A N F action on physiologically identified D M N neurons
Eight male Long Evans rats were anesthetized with urethane and tracheotomized as described. Bipolar stimulation electrodes 3 were attached to the cervical vagus nerve for the purpose of antidromically activating DMN neurons. Animals were mounted in the stereotaxic frame and the dorsal medulla was exposed. Triplebarrelled glass recording/injection pipettes 2° were placed in a hydraulic mierodrive unit (Kopf 650, Tujunga, CA). The recording barrel contained 2 M NaCI + 2% pontamine Sky blue. The injection barrels contained either atriopeptin III (0.5 mM) in isotonic saline or isotonic saline. Electrical signals from the recording barrel were amplified, displayed on storage oscilloscopes and recorded on magnetic tape. Amplified signals were also processed in real time by a window discriminator/rate meter circuit which produced plots of integrated firing rate vs time. Taped records were also analysed using the RC Electronics waveform processing system. The pipette array was advanced into the dorsal medulla while twin stimulation pulses (100-1000/~A, 0.5 ms, 50-100 Hz) were applied to the vagus nerve. Single units that respond (a) with a fixed latency after stimulation, (b) to high frequency (50-100 Hz) stimulation, (c) with collision to spontaneous action potentials, were considered to be antidromically activated by vagal stimulation 9'2°'21. Such units were tested for a response to micropressure injections of either isotonic saline or ANF solutions. Injection volumes were measured as described above. At the end of the study, eathodal direct current (25/~A for 20 min) was applied to the recording barrel to eject pontamine dye into the recording site. Animals were then perfused with formalin and their brainstems were removed. The tissue was fixed in formalin for 24 h, and then sectioned, mounted, counterstained and examined for the location of the injected pontamine dye. RESULTS
b
C lg
4OOs U L
"0 t
20..
d i
VEH
:
ANF
10_ :
i
O. Ii
t'-
"-
-- 10_
•~ o
--20_
E -3O ¢-
ANF
(rat
atriopeptin
III)
produced
a significant
r e d u c t i o n in gastric motility w h e n a d m i n i s t e r e d into the D M N . I n j e c t i o n s of 20 p m o l o f this p e p t i d e d e c r e a s e d the motility i n d e x by a p p r o x i m a t e l y 3 0 % , ( Z = 2.36, P < 0.02, W i l c o x o n test; see Fig. 1). F u r t h e r m o r e , in 5 of 7 animals, a r e d u c t i o n in gastric s m o o t h muscle tension (i.e. gastric t o n e ) was o b s e r v e d after A N F a d m i n i s t r a t i o n ( a v e r a g e r e d u c t i o n = 260 + 9 mg). T h e antimotility effects o f A N F w e r e not a p p a r e n t in rats that w e r e v a g o t o m i z e d (post v a g o t o m y 5 min motility index b e f o r e
E -4o
if)
Fig. 1. Effect on gastric motility and tone of DMN injections of vehicle (20 pl of isotonic saline; panel a) or ANF (20 pmol in 20 ni; panel b). In a different animal, a cervical vagotomy was performed 30 min prior to ANF administration into the DMN (panel c). Panel d is a data summary (n = 7) showing the effects of vehicle (VEH) and atrial natriuretic factor (ANF) on the difference in the motility index measured before and after injection. DMN injections of ANF produced a significant reduction in motility (* = P < 0.02; Wilcoxon test) in intact, but not vagotomized animals (ANF/VAGOX; n = 4). Abbreviation: g, gram of tension.
249
a
v400pl
ANF 25fmol x 50pl
10-PPS 0 ~tLl,,,~,,,,,I,,,,,,,,,,,l~,,,,aJJJ,,I,,
,4,~,~,~lt,,,~,~,t,,I,,,,J~ ~,,,,I,~I/,~,~L,I,L,~,,,,~,,I
0
b
8min
¢
20 ms
20 ms
Fig. 2. Panel a is a record of the integrated firing frequency (PPS = pulses per s) of a physiologically identified DMN neuron after pressure injection of vehicle (V) (400 pl) and ANF (25 fmol in 50 pl). Panels b and c represent the criteria used to identify this cell as a DMN neuron. The 10 superimposed oscillograph traces in panel b illustrate the constancy of the response to cervical vagal stimulation (arrows represent stimulus artifact; stars represent action potentials). Also note that this neuron responded to high frequency stimulation (65 Hz) of the vagus nerve. In panel c, a spontaneously occurring action potential (white arrow) 'collided' with the first antidromic potential (dark arrow). As a result, only the second stimulation-induced action potential (star) is apparent.
were excited by A N F (dose range = 25-500 fmol), none were inhibited (see Figs. 2 and 3). The remaining 5 neurons did not respond to the administration of this peptide. The site of a D M N neuron that was excited by A N F is illustrated in Fig. 4. DISCUSSION The findings in the present study demonstrate that ANF, when injected into the D M N , can influence the vagal control of gastric function. Gastric motility and tone were significantly reduced by D M N injections of this peptide, an effect that was abolished by bilateral vagotomy. The electrophysiological data provide additional support for the hypothesis that A N F can modulate the activity of vagal efferent neurons. The firing rates of D M N cells that were responsive to A N F were increased by this peptide. Electrical stimulation of vagal efferents produces an excitation, superimposed on an inhibition of gastric motor function 1,1°.19,24. This is due to the fact that vagal
preganglionic neurons contact neurons in the myenteric plexus of the stomach 4' 17; myenteric neurons can increase or decrease gastric motor activity 1°. Our data suggest that A N F activates vagal preganglionic neurons that synapse with inhibitory neurons in the myenteric plexus to produce an inhibition of gastric motility and smooth muscle tension. In contrast to gastric function, heart rate was not changed by the same injection of A N F into the DMN. This suggests that the vagal efferents to the heart were uneffected by A N E These data confirm reports demonstrating that A N F has no effect on heart rate or blood pressure when injected into the D M N 8'2z. Also consistent with these findings is the fact that the spontaneous firing rates of 1/3 of the D M N neurons were not changed by administration of A N F ; perhaps these D M N neurons projected to targets that are not influenced by D M N administration of ANF, such as the heart. Taken together, these results suggest that A N F selectively activates a subset of vagal neurons projecting to inhibitory enteric circuits innervating gastric smooth muscle, but not
250
!L..... L ~ , , . J
.... . ~ . . ~
A
v
A
B r ~
r
C V
A
Fig. 3. Panel A illustrates the antiomic activation of a DMN neuron. Note the constant latency to respond to high frequency (65 Hz) vagal nerve stimulation. Panel B is an integrated firing frequency plot of the same neuron depicted in panel A, in response to vehicle (V = 200 pl) and ANF (A = 100 fmol in 200 pl). The oscillograph records shown in panel C demonstrate the increased activity of this unit in response to A N F administration.
Fig. 4. Coronal section of the brainstem showing the location of a pontamine blue dye spot, which was ejected from the recording barrel to mark the recording site where an ANF-responsive DMN cell was found. Abbreviations: AP, area postrema; CC, central canal; DMN, dorsal motor nucleus; NTS, nucleus of the solitary tract.
251 those projecting to the heart. This supports the view that central peptidergic pathways select 'visceral m o t o r programs' by acting on different populations of vagal efferent neurons. Perhaps this is accomplished by virtue of the existence of a differential distribution of peptide receptors on the vagal preganglionic neurons; this would allow various peptidergic CNS pathways to modulate selectively the activity of vagal targets. The origin of the A N F projection to the dorsal vagal complex has not yet been established. A N F - c o n t a i n i n g p e r i k a r y a are located in n u m e r o u s sites in the CNS, including several which are known to maintain a direct connection with the D M N , i.e. the bed nucleus of the stria terminalis (BNST), the central nucleus of the a m y g d a l a ( C N A ) and the paraventricular nucleus of the h y p o t h a l a m u s (PVN) 14"31"34. W h e r e a s electrical stimulation of B N S T or C N A elevated gastric motility 12'~3, activation of the P V N p r o d u c e d a complex, excitatory followed by an inhibitory effect on gastric motility. The inhibitory c o m p o n e n t was effectively blocked by an antagonist to oxytocin 29, which is a putative transmitter in a p o r t i o n of the neurons projecting from the PVN to
the D M N . A N F has been r e p o r t e d to be co-localized with oxytocin (OT) in P V N and supraoptic nucleus 15. Al-
REFERENCES
621-683. 12 Hermann, G.E., McCann, M.J. and Rogers, R.C., Activation of the bed nucleus of the stria terminalis increases gastric motility in the rat, J. Auton. Nerv. Sys., 30 (1990) 123-128. 13 Hermann, G.E. and Rogers, R.C., Extrinsic neural control of brainstem gastric vagovagal reflex circuits. In M.V. Singer and H. Goebell (Eds.), Nerves and the Gastrointestinal Tract, Kluwer Academic Publishers, Boston, MA, 1989, pp. 345-364. 14 Holstege, G., Meiners, L. and Tan, K., Projections of the bed nucleus of the stria terminalis to the mesencephalon, ports and medulla obiongata in the cat, Exp. Brain Res., 58 (1985) 379-391. 15 Jirikowski, G.E, Back, H., Forssmann, W.G. and Stumpf, W.E., Coexistence of atrial natriuretic factor (ANF) and oxytocin in neurons of the rat hypothalamus, Neuropeptides, 8 (1986) 243-249. 16 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 17 Kirchgessner, A.L. and Gershon, M.D. Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat, J. Comp. Neurol., 285 (1989) 38-53. 18 Laughton, W.B. and Powley, T.L., Localization of efferent function in the dorsal motor nucleus of the vagus, Am. J. Physiol., 252 (1987) R13-R25. 19 Martinson, J. and Muren, A., Excitatory and inhibitory effects of vagal stimulation on gastric motility in the cat, Acta Physiol. Scand., 57 (1963) 309-316. 20 McCann, M.J. and Rogers, R.C., Thyrotropin releasing hormone: effects on identified neurons of the dorsal vagal complex, J. Auton. Nerv. Sys., 26 (1989) 107-112. 21 McCann, M.J. and Rogers, R.C., Oxytocin excites gastricrelated neurones in rat dorsal vagal complex, J. Physiol., 428 (1990) 95-108. 22 McKitrick, D.J. and Calaresu, ER., Cardiovascular responses to microinjection of ANF into dorsal medulla of rats, Am. J. Physiol., 255 (1988) R182-R187. 23 Nilaver, G., Rodenbaum, L.C., Fukui, K., Neuwelt, E.A.,
1 Abrahamsson, H., Studies on the inhibitory nervous control of gastric motility, Acta Physiol. Scand., Suppl. 390 (1973) 1-38. 2 Atlas, S.A., Kleinert, H.D., Camargo, M.J., Januszewicz, A., Sealey, J.E., Laragh, J.H., Shilling, J.W., Lewicki, J.A., Johnson, L.K. and Maak, T., Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide, Nature, 309 (1984) 717-719. 3 Barone, EC., Wayner, M.J., Aquilar-Baturoni, H.V. and Guevara-Aquilar, R.A., A bipolar electrode for peripheral nerve stimulation, Brain Res. Bull., 4 (1979) 421-422. 4 Berthoud, H.R., Jedrzejewska, A. and Powley, T.L. Simultaneous identification of afferent inputs and efferent projections of the dorsal motor nucleus, Soc. Neurosci. Abstr., 15 (1989) 264. 5 Currie, M.G., Geller, D.M., Cole, B.R., Boylan, J.G., Yu Shang, W., Holmberg, S.W. and Needleman, P., Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria, Science, 221 (1984) 67-69. 6 De Bold, A.J., Atrial natriuretic factor of the heart. Studies on isolation and properties, Proc. Soc. Exp. Biol. Med., 170 (1982) 133-138. 7 De Bold, A.J., Atrial natriuretic factor: an overview, Fed. Proc., 45 (1986) 2081-2084. 8 Ermirio, R., Ruggeri, P, Cogo, C.E., Molinari, C. and Calaresu, ER., Neuronal and cardiovascular responses to ANF microinjected into the solitary nucleus, Am. J. Physiol., 256 (1989) R577-R582. 9 Fuller. J.H. and Schlag, J.D., Determination of antidromic excitation by the collision test; problems of interpretation, Brain Research, 112 (1976) 283-298. 10 Gillespie, J.S. Non-adrenergic non-cholinergic inhibitory control of gastrointestinal motility. In M. Weinbeck (Ed.), Motility in the Digestive Tract, Raven Press, New York, 1982, pp. 51-66. 11 Gillis, R.A., Quest, J.A., Pagani, ED. and Norman, W.P., Control centers in the central nervous system for regulating gastrointestinal motility. In J.D. Wood (Ed.), Handbook of Physiology, Gastrointestinal Physiology, Sec. 6, Vol. 1, Pt. 1, American Physiological Society, Bethesda, MD, 1989, pp.
though it is not known if O T and A N F coexist in the PVN projection to the D M N , it is interesting to note that D M N injections of OT, like A N F , result in an inhibition of gastric motility via activation of vagal efferents that connect with inhibitory enteric neurons 21'29. In contrast to A N F , however, O T does influence vagal control of the heart, as indicated by a reduction of heart rate when O T is administered into the D M N 28. Nonetheless, the coexistence of these two peptides will require additional verification because of a recent r e p o r t showing that some A N F polyclonal antibodies cross-react with neurophysin I and II, the carrier molecules for oxytocin and vasopressin 23. In conclusion, our results point to a n o t h e r CNS action of A N F as an ' A n t i - M o t i l i t y F a c t o r ' which activates vagal efferents that connect with inhibitory enteric pathways in the stomach. Acknowledgements. This work was supported in part by NINCDS Grants NS24530 and NS08690 to R.C.R. and M.J.M.
252
24
25 26
27
28
29
Samson, W.K., Zimmerman, E.A. and Gibbs, D.M., An antiserum to atrial natriuretic factor (ANF) cross-reacts with neurophysins in the hypothalamo-neurohypophysial system of rat brain, Neuropeptides, 14 (1989) 137-144. Ohta, T., Nakazato and Ohga, A., Reflex control of gastric motility by the vagus and splanchnic nerves in the guinea pig in vivo, J. Auton. Nerv. Sys., 14 (1985) 137-149. Ormsbee, H.S. and Bass, P., Gastroduodenal motor gradients in the dog after pyloroplasty, Am. J. Physiol., 230 (1976) 389-397. Pagani, ED., Norman, W.P., Kasbekar, D.K. and Gillis, R.A., Localization of sites within dorsal motor nucleus of the vagus that affect gastric motility, Am. J. Physiol., 249 (1985) G73-G84. Quirion, R., Dalpe, M. and Dam, T.-V., Characterization and distribution of receptors for the atrial natriuretic peptides in mammalian brain, Proc. Natl. Acad. Sci., 83 (1986) 174-178. Rogers, R.C. and Hermann, G.E., Dorsal medullary oxytocin, vasopressin, oxytocin antagonist and TRH effects on gastric acid secretion and heart rate, Peptides, 6 (1985) 1143-1148. Rogers, R.C. and Hermann, G.E., Oxytocin, oxytocin antagonist, TRH and hypothalamic paraventricular nucleus stimulation effects on gastric motility, Peptides, 8 (1987) 505-513.
30 Saper, C.B., Standaert, D.G., Currie, M.G., Schwartz, D., Geller, D.M. and Needleman, P., Atriopeptin-immunoreactive neurons in the brain: Presence in cardiovascular regulatory areas, Science, 227 (1985) 1047-1049. 31 Schwaber, J.S., Kapp, B.S., Higgins, G.A. and Rapp, P.R., Amygdaloid and basal forebrain connections with the nucleus of the solitary tract and the dorsal motor nucleus, J. Neurosci., 2 (1982) 1424-1438. 32 Skofitsch, G. and Jacobowitz, D.M., Atrial natriuretic peptide in the central nervous system of the rat, Cell. Mol. Neurobiol., 8 (1988) 339-391. 33 Standaert, D.G., Needleman, P. and Saper, C. Organization of atriopeptin-like immunoreactive neurons in the central nervous system, J. Comp. Neurol., 253 (1986) 315-341. 34 Swanson, L.W. and Kuypers, H.G.J.M., The parventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and the organization of projections to the pituitary, the dorsal vagal complex and spinal cord as demonstrated by retrograde fluorescence double-labelling methods, J. Comp. Neurol., 194 (1980) 555-570.
Brain Research, 549 (1991) 247-252 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116613F BRES 16613
Dorsal medullary injection of atrial natriuretic factor (ANF) excites vagal efferents and inhibits gastric motility Monica J. McCann, Kathy Nice-Lepard and Richard C. Rogers Department of Physiology and Neuroscience Program, Ohio State University College of Medicine, Columbus, OH 43210 (U.S.A.) (Accepted 11 December 1990)
Key words: Atrial natriuretic factor; Medulla oblongata; Gastric motility; Efferent vagus; Extracellular recording
Atrial natriuretic factor (ANF)-immunoreactive fibers are found in the dorsal motor nucleus of the vagus (DMN) along with receptors for that peptide. Previous investigations showed that ANF injections into the DMN did not influence cardiovascular functions. Since the DMN is largely (but not exclusively) involved with the control of gastrointestinal functions, we hypothesized that ANF may act on gastric, rather than cardiovascular vagal efferents. Injections of ANF (20 pmol rat atriopeptin III in 20 nl) into the DMN evoked a vagally dependent reduction in gastric motility. In a separate electrophysiologicai study, 10 of 15 (66%) antidromically identified DMN neurons were excited by micropressure-applied ANF (25-500 fmol in 50-1000 pl). We propose that ANF-containing neurons in the DMN reduce gastric motility by activating vagal efferents that synapse with inhibitory neurons in the gastric enteric nervous system. INTRODUCTION
using a combination of micropressure injection and extracellular recording techniques.
Atrial natriuretic factor ( A N F ) , which was first isolated from extracts of rat atria, is known to have n u m e r o u s effects on cardiovascular function and fluid homeostasis when administered into the peripheral circulation2"5-7. Immunocytochemical studies report that A N F is also present within n e u r o n s in the central nervous system ~6'3°'32'33. The role(s) of A N F in the CNS remains to be elucidated, however it is interesting to note that ANF-like immunoreactive fibers and high affinity A N F binding sites are found in the nucleus of the solitary tract (NTS) and the dorsal motor nucleus (DMN) 16"27'32,33. This suggests that A N F may influence the vagal control of the heart and/or other vagally innervated organs. Indeed, several investigators have reported that A N F injections into the NTS provoke a modest (5%) cardioacceleration while similar injections into the D M N have no effect on cardiovascular function8,22. Because the D M N also is involved in the parasympathetic control of digestive functions H.18,26, we hypothesized that A N F may influence vagal efferent outflow to the stomach. We investigated this hypothesis by measuring the effect of A N F injections into the D M N on gastric corpus/antrum motility as well as heart rate in the rat. Furthermore, in a second set of experiments, we determined whether A N F influenced the firing rate of single D M N neurons
MATERIALS AND METHODS
ANF/gastric motility studies Male Long-Evans rats (250-450 g) were anesthetized with urethane (ethylcarbamate, 1.2 g/kg). The trachea was catheterized to maintain an open airway. Then, a laparotomy was performed and a miniature strain gauge (RB Products, 153 Nautilus Dr. Madison, WI) was sewn in parallel with corpus/antral circular smooth muscle to measure gastric motility and tone. After closing the incision, the animal was mounted in a stereotaxic frame, and the dorsal medulla was exposed. The tips of combination metal microstimulation/ double barrelled micropipette arrays2s were placed in the medial portion of the DMN. One pipette barrel was filled with ANF (rat atriopeptin III; ANF 5-28, Sigma, St. Louis, MO) dissolved in isotonic saline while the other barrel contained only the saline solution. Both pipette barrels were connected to a micropressure injection system (Medical Systems BH2, Great Neck, NY). The metal electrode was used to determine when the electrode array was positioned in the DMN. Briefly, electrical pulses (25/tA, 0.5 ms duration, 10 Hz, 2-5 s) were generated by a conventional stimulator and delivered to the electrode through a constant-current stimulus isolation unit; this stimulus results in a reduction in heart rate and gastric motility when the tip is positioned in the DMN29. The location of the pipette tips were confirmed by passing 50 pA cathodal d.c. through the stimulating electrode. Lesions were subsequently located by examining the brainstem histologically. To measure gastric motility the leads from the strain gauge were connected to a Wheatstone bridge-based strain gauge amplifier of conventional design. The output of this amplifier was directed simultaneously to a Grass polygraph and to the A-to-D convertor inputs of an RC Electronics waveform storage/analysis system. In
Correspondence: M.J. McCann, Department of Physiology, Ohio State University College of Medicine, 333 W. 10th Ave., Columbus, OH 43210, U.S.A.
248 this way, gastric motility data could be displayed in real time while being digitized for subsequent analysis or graphic display. To measure the heart rate, needle electrodes were placed over the thorax and hindlimb to record the ECG. ECG signals were used to produce a continuous plot of heart rate vs time using a ratemeter circuit connected to another channel of the polygraph. While monitoring motility and heart rate, either saline (20 nl) or ANF (20 pmol in 20 nl) was injected into the DMN (n = 7 rats). The amount of solution ejected from the pipettes was measured directly by observing the meniscus of the ejectate in the pipette with a 175 × long-working distance microscope (Melles Griot, Irvine, CA). Each injection was separated in time by at least 20 min. In an additional group of 4 rats, suture loops were placed around the vagal trunks after catheterizing the trachea. Thirty min prior to the administration of ANF into the DMN, the vagus nerves were severed by pulling the suture loops. All motility data were scored by applying the minute motility index 25 for a period of 5 min before and 5 min after DMN injections of saline or ANE Non-parametric statistics (Wilcoxon matched pairs test) were applied to the differences in motility indices between saline and ANF treatments.
A N F = 23.3 + 2.0 ( S . E . M . ) ; i n d e x after A N F = 22.0 _+ 2.5, P > 0.05; see Fig. l c and d). In c o n t r a s t to gastric motility, h e a r t rate was not a l t e r e d by A N F injections c e n t e r e d on the D M N
(data not shown).
Histological
analysis c o n f i r m e d the p o s i t i o n of the p i p e t t e tips within the D M N . In the s e c o n d series of e x p e r i m e n t s , A N F was microp r e s s u r e - i n j e c t e d on D M N n e u r o n s that w e r e identified using e l e c t r o p h y s i o l o g i c a l criteria. O f 28 a n t i d r o m i c a i l y a c t i v a t e d cells e n c o u n t e r e d , 15 cells w e r e r e c o r d e d for a sufficient length o f t i m e to d e t e r m i n e its r e s p o n s e to i n j e c t i o n of saline v e h i c l e and A N E O f t h e s e 15 cells, 10
A N F action on physiologically identified D M N neurons
Eight male Long Evans rats were anesthetized with urethane and tracheotomized as described. Bipolar stimulation electrodes 3 were attached to the cervical vagus nerve for the purpose of antidromically activating DMN neurons. Animals were mounted in the stereotaxic frame and the dorsal medulla was exposed. Triplebarrelled glass recording/injection pipettes 2° were placed in a hydraulic mierodrive unit (Kopf 650, Tujunga, CA). The recording barrel contained 2 M NaCI + 2% pontamine Sky blue. The injection barrels contained either atriopeptin III (0.5 mM) in isotonic saline or isotonic saline. Electrical signals from the recording barrel were amplified, displayed on storage oscilloscopes and recorded on magnetic tape. Amplified signals were also processed in real time by a window discriminator/rate meter circuit which produced plots of integrated firing rate vs time. Taped records were also analysed using the RC Electronics waveform processing system. The pipette array was advanced into the dorsal medulla while twin stimulation pulses (100-1000/~A, 0.5 ms, 50-100 Hz) were applied to the vagus nerve. Single units that respond (a) with a fixed latency after stimulation, (b) to high frequency (50-100 Hz) stimulation, (c) with collision to spontaneous action potentials, were considered to be antidromically activated by vagal stimulation 9'2°'21. Such units were tested for a response to micropressure injections of either isotonic saline or ANF solutions. Injection volumes were measured as described above. At the end of the study, eathodal direct current (25/~A for 20 min) was applied to the recording barrel to eject pontamine dye into the recording site. Animals were then perfused with formalin and their brainstems were removed. The tissue was fixed in formalin for 24 h, and then sectioned, mounted, counterstained and examined for the location of the injected pontamine dye. RESULTS
b
C lg
4OOs U L
"0 t
20..
d i
VEH
:
ANF
10_ :
i
O. Ii
t'-
"-
-- 10_
•~ o
--20_
E -3O ¢-
ANF
(rat
atriopeptin
III)
produced
a significant
r e d u c t i o n in gastric motility w h e n a d m i n i s t e r e d into the D M N . I n j e c t i o n s of 20 p m o l o f this p e p t i d e d e c r e a s e d the motility i n d e x by a p p r o x i m a t e l y 3 0 % , ( Z = 2.36, P < 0.02, W i l c o x o n test; see Fig. 1). F u r t h e r m o r e , in 5 of 7 animals, a r e d u c t i o n in gastric s m o o t h muscle tension (i.e. gastric t o n e ) was o b s e r v e d after A N F a d m i n i s t r a t i o n ( a v e r a g e r e d u c t i o n = 260 + 9 mg). T h e antimotility effects o f A N F w e r e not a p p a r e n t in rats that w e r e v a g o t o m i z e d (post v a g o t o m y 5 min motility index b e f o r e
E -4o
if)
Fig. 1. Effect on gastric motility and tone of DMN injections of vehicle (20 pl of isotonic saline; panel a) or ANF (20 pmol in 20 ni; panel b). In a different animal, a cervical vagotomy was performed 30 min prior to ANF administration into the DMN (panel c). Panel d is a data summary (n = 7) showing the effects of vehicle (VEH) and atrial natriuretic factor (ANF) on the difference in the motility index measured before and after injection. DMN injections of ANF produced a significant reduction in motility (* = P < 0.02; Wilcoxon test) in intact, but not vagotomized animals (ANF/VAGOX; n = 4). Abbreviation: g, gram of tension.
249
a
v400pl
ANF 25fmol x 50pl
10-PPS 0 ~tLl,,,~,,,,,I,,,,,,,,,,,l~,,,,aJJJ,,I,,
,4,~,~,~lt,,,~,~,t,,I,,,,J~ ~,,,,I,~I/,~,~L,I,L,~,,,,~,,I
0
b
8min
¢
20 ms
20 ms
Fig. 2. Panel a is a record of the integrated firing frequency (PPS = pulses per s) of a physiologically identified DMN neuron after pressure injection of vehicle (V) (400 pl) and ANF (25 fmol in 50 pl). Panels b and c represent the criteria used to identify this cell as a DMN neuron. The 10 superimposed oscillograph traces in panel b illustrate the constancy of the response to cervical vagal stimulation (arrows represent stimulus artifact; stars represent action potentials). Also note that this neuron responded to high frequency stimulation (65 Hz) of the vagus nerve. In panel c, a spontaneously occurring action potential (white arrow) 'collided' with the first antidromic potential (dark arrow). As a result, only the second stimulation-induced action potential (star) is apparent.
were excited by A N F (dose range = 25-500 fmol), none were inhibited (see Figs. 2 and 3). The remaining 5 neurons did not respond to the administration of this peptide. The site of a D M N neuron that was excited by A N F is illustrated in Fig. 4. DISCUSSION The findings in the present study demonstrate that ANF, when injected into the D M N , can influence the vagal control of gastric function. Gastric motility and tone were significantly reduced by D M N injections of this peptide, an effect that was abolished by bilateral vagotomy. The electrophysiological data provide additional support for the hypothesis that A N F can modulate the activity of vagal efferent neurons. The firing rates of D M N cells that were responsive to A N F were increased by this peptide. Electrical stimulation of vagal efferents produces an excitation, superimposed on an inhibition of gastric motor function 1,1°.19,24. This is due to the fact that vagal
preganglionic neurons contact neurons in the myenteric plexus of the stomach 4' 17; myenteric neurons can increase or decrease gastric motor activity 1°. Our data suggest that A N F activates vagal preganglionic neurons that synapse with inhibitory neurons in the myenteric plexus to produce an inhibition of gastric motility and smooth muscle tension. In contrast to gastric function, heart rate was not changed by the same injection of A N F into the DMN. This suggests that the vagal efferents to the heart were uneffected by A N E These data confirm reports demonstrating that A N F has no effect on heart rate or blood pressure when injected into the D M N 8'2z. Also consistent with these findings is the fact that the spontaneous firing rates of 1/3 of the D M N neurons were not changed by administration of A N F ; perhaps these D M N neurons projected to targets that are not influenced by D M N administration of ANF, such as the heart. Taken together, these results suggest that A N F selectively activates a subset of vagal neurons projecting to inhibitory enteric circuits innervating gastric smooth muscle, but not
250
!L..... L ~ , , . J
.... . ~ . . ~
A
v
A
B r ~
r
C V
A
Fig. 3. Panel A illustrates the antiomic activation of a DMN neuron. Note the constant latency to respond to high frequency (65 Hz) vagal nerve stimulation. Panel B is an integrated firing frequency plot of the same neuron depicted in panel A, in response to vehicle (V = 200 pl) and ANF (A = 100 fmol in 200 pl). The oscillograph records shown in panel C demonstrate the increased activity of this unit in response to A N F administration.
Fig. 4. Coronal section of the brainstem showing the location of a pontamine blue dye spot, which was ejected from the recording barrel to mark the recording site where an ANF-responsive DMN cell was found. Abbreviations: AP, area postrema; CC, central canal; DMN, dorsal motor nucleus; NTS, nucleus of the solitary tract.
251 those projecting to the heart. This supports the view that central peptidergic pathways select 'visceral m o t o r programs' by acting on different populations of vagal efferent neurons. Perhaps this is accomplished by virtue of the existence of a differential distribution of peptide receptors on the vagal preganglionic neurons; this would allow various peptidergic CNS pathways to modulate selectively the activity of vagal targets. The origin of the A N F projection to the dorsal vagal complex has not yet been established. A N F - c o n t a i n i n g p e r i k a r y a are located in n u m e r o u s sites in the CNS, including several which are known to maintain a direct connection with the D M N , i.e. the bed nucleus of the stria terminalis (BNST), the central nucleus of the a m y g d a l a ( C N A ) and the paraventricular nucleus of the h y p o t h a l a m u s (PVN) 14"31"34. W h e r e a s electrical stimulation of B N S T or C N A elevated gastric motility 12'~3, activation of the P V N p r o d u c e d a complex, excitatory followed by an inhibitory effect on gastric motility. The inhibitory c o m p o n e n t was effectively blocked by an antagonist to oxytocin 29, which is a putative transmitter in a p o r t i o n of the neurons projecting from the PVN to
the D M N . A N F has been r e p o r t e d to be co-localized with oxytocin (OT) in P V N and supraoptic nucleus 15. Al-
REFERENCES
621-683. 12 Hermann, G.E., McCann, M.J. and Rogers, R.C., Activation of the bed nucleus of the stria terminalis increases gastric motility in the rat, J. Auton. Nerv. Sys., 30 (1990) 123-128. 13 Hermann, G.E. and Rogers, R.C., Extrinsic neural control of brainstem gastric vagovagal reflex circuits. In M.V. Singer and H. Goebell (Eds.), Nerves and the Gastrointestinal Tract, Kluwer Academic Publishers, Boston, MA, 1989, pp. 345-364. 14 Holstege, G., Meiners, L. and Tan, K., Projections of the bed nucleus of the stria terminalis to the mesencephalon, ports and medulla obiongata in the cat, Exp. Brain Res., 58 (1985) 379-391. 15 Jirikowski, G.E, Back, H., Forssmann, W.G. and Stumpf, W.E., Coexistence of atrial natriuretic factor (ANF) and oxytocin in neurons of the rat hypothalamus, Neuropeptides, 8 (1986) 243-249. 16 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 17 Kirchgessner, A.L. and Gershon, M.D. Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat, J. Comp. Neurol., 285 (1989) 38-53. 18 Laughton, W.B. and Powley, T.L., Localization of efferent function in the dorsal motor nucleus of the vagus, Am. J. Physiol., 252 (1987) R13-R25. 19 Martinson, J. and Muren, A., Excitatory and inhibitory effects of vagal stimulation on gastric motility in the cat, Acta Physiol. Scand., 57 (1963) 309-316. 20 McCann, M.J. and Rogers, R.C., Thyrotropin releasing hormone: effects on identified neurons of the dorsal vagal complex, J. Auton. Nerv. Sys., 26 (1989) 107-112. 21 McCann, M.J. and Rogers, R.C., Oxytocin excites gastricrelated neurones in rat dorsal vagal complex, J. Physiol., 428 (1990) 95-108. 22 McKitrick, D.J. and Calaresu, ER., Cardiovascular responses to microinjection of ANF into dorsal medulla of rats, Am. J. Physiol., 255 (1988) R182-R187. 23 Nilaver, G., Rodenbaum, L.C., Fukui, K., Neuwelt, E.A.,
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though it is not known if O T and A N F coexist in the PVN projection to the D M N , it is interesting to note that D M N injections of OT, like A N F , result in an inhibition of gastric motility via activation of vagal efferents that connect with inhibitory enteric neurons 21'29. In contrast to A N F , however, O T does influence vagal control of the heart, as indicated by a reduction of heart rate when O T is administered into the D M N 28. Nonetheless, the coexistence of these two peptides will require additional verification because of a recent r e p o r t showing that some A N F polyclonal antibodies cross-react with neurophysin I and II, the carrier molecules for oxytocin and vasopressin 23. In conclusion, our results point to a n o t h e r CNS action of A N F as an ' A n t i - M o t i l i t y F a c t o r ' which activates vagal efferents that connect with inhibitory enteric pathways in the stomach. Acknowledgements. This work was supported in part by NINCDS Grants NS24530 and NS08690 to R.C.R. and M.J.M.
252
24
25 26
27
28
29
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30 Saper, C.B., Standaert, D.G., Currie, M.G., Schwartz, D., Geller, D.M. and Needleman, P., Atriopeptin-immunoreactive neurons in the brain: Presence in cardiovascular regulatory areas, Science, 227 (1985) 1047-1049. 31 Schwaber, J.S., Kapp, B.S., Higgins, G.A. and Rapp, P.R., Amygdaloid and basal forebrain connections with the nucleus of the solitary tract and the dorsal motor nucleus, J. Neurosci., 2 (1982) 1424-1438. 32 Skofitsch, G. and Jacobowitz, D.M., Atrial natriuretic peptide in the central nervous system of the rat, Cell. Mol. Neurobiol., 8 (1988) 339-391. 33 Standaert, D.G., Needleman, P. and Saper, C. Organization of atriopeptin-like immunoreactive neurons in the central nervous system, J. Comp. Neurol., 253 (1986) 315-341. 34 Swanson, L.W. and Kuypers, H.G.J.M., The parventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and the organization of projections to the pituitary, the dorsal vagal complex and spinal cord as demonstrated by retrograde fluorescence double-labelling methods, J. Comp. Neurol., 194 (1980) 555-570.
